The goal of this proposal is to comprehensively map and identify the subnetworks of synaptic protein complexes that are central players in the synaptic dysfunction occurring with neurodegeneration. We will use the emerging power of quantitative network proteomics in the Emili laboratory to systematically characterize the major protein assemblies present at normal and diseased synapses on a proteome scale. This research will be propelled by discoveries from the Wolozin laboratory demonstrating that a dynamic network of protein interactions drives tau biology and changes with the course of disease. Interpreting these perturbed assembly networks, though, demands knowledge of the localization and compositional specificity of such complexes. The unbiased interactome screening technology developed by the Emili laboratory is uniquely suited for unbiased interrogations of synaptic protein networks. We hypothesize that selective disruption of specific synaptic protein assemblies mediates the functional degeneration associated with tauopathy. Aim 1 will determine how synaptic protein complexes differ between general cortical and cholinergic neurons. We will isolate and biochemical separate synaptic assemblies from total cortical and cholinergic (ChAT::GFP) synapses, using FACS to further purify ChAT:GFP synapses. Separated assemblies will be characterized by precision mass spectrometry and integrative data mining (machine learning) procedures to determine their composition and post- translational modification states, and to map dynamically changing interactions implicated in altered synaptic function during normal aging. Aim 2 will determine how the macromolecular structures of synaptic protein assemblies change with aging and AD. We will analyze cortical synaptosomal complexes from P301S tau and P301S tau x TIA1+/- mice, the latter which exhibited delayed degeneration. Key drivers in synaptic dysfunction will be identified and verified in AD and control human samples by co-immunoprecipitation. Aim 3 will identify complexes that are critical drivers of synaptic function by disrupting prioritized assemblies using genetic and opticogenetic tools. This work will determine key regulators of synaptic function in health and disease, and will also produce expanded genetic tools and outstanding targets for future approaches using bio-engineered regulation.